Project Summary Zika virus (ZIKV), a mosquito-borne flavivirus, was originally discovered in Uganda in 1947, and for nearly all of its history has been associated with mild self-limited illness. This changed during recent and ongoing ZIKV outbreaks in the South Pacific and Latin America, which have revealed new modes of transmission (i.e. sexual and in utero), as well as unexpected disease presentations (including birth defects and Guillain-Barr syndrome). These severe manifestations of ZIKV infection all involve the virus invading tissues that ordinarily are protected by anatomic barriers, including the central nervous system, the testes, and the fetal compartment. Understanding the viral and host factors that determine ZIKV tissue tropism is critical for evaluating the pathogenic potential of emerging ZIKV strains in new host environments. In this proposal, we will employ a new ZIKV reverse genetics system to experimentally test the hypothesis that genetic changes in the ZIKV strains currently circulating in Latin America confer enhanced virulence. In particular, we will focus on N-linked glycosylation in the virion envelope protein, as this is well-established as a virulence determinant in other flaviviruses and differs between contemporary outbreak strains of ZIKV and historical ones. Since novel tissue tropism may contribute to new manifestations of ZIKV infection, we will investigate the role of interferon lambda (IFN-?) in maintaining barriers to ZIKV tissue tropism, including the blood-brain barrier, placenta, and sertoli cell barrier, as has been shown previously for West Nile virus neuroinvasion. We will infect mice that lack the IFN-L receptor and compare ZIKV viral loads in the central nervous system, fetal compartment, testes, and eyes to tissues that are not protected by specialized barriers and to wild-type mice. Conversely, we will infect mice that lack the IFN-ab receptor, which develop high viral loads and tissue dissemination, with recombinant IFN-L protein and determine whether this results in barrier tightening and restriction of ZIKV invasion. As IFN-L protein has been used successfully in phase II clinical trials as a therapy for hepatitis C virus infection, this approach would provide a potential method to treat severe ZIKV infection. Altogether, our studies will reveal key viral and host determinants of ZIKV tissue tropism, key aspects of understanding the severe manifestations of ZIKV infection observed in the current outbreak in Latin America.